Mass spectrometry is used to measure the mass of a sample molecule, as well as the mass of the fragments of a sample to identify that sample. The simplest mass spectrometers introduce a gaseous, electrically neutral sample in vacuo, normally at pressures of 10.sup.-6 torr or less. Silverstein, et al, Spectrometric Identification of Organic Compounds, p.7 (John Wiley & Sons, Inc. 1963). The sample then passes through an electron beam.
The fast-moving electrons from the electron beam strike electrons on the sample being studied, ejecting one or more electrons from the sample. After a subject sample molecule has lost an electron, the sample has a net positive charge, or is "ionized."
Mass spectrometry measures the ratio of the mass of the molecule to the ion's electric charge. The mass is customarily expressed in terms of atomic mass units, called Daltons. The charge or ionization is customarily expressed in terms of multiples of elementary charge. The ratio of the two is expressed as a m/z ratio value (mass/charge or mass/ionization ratio). Because the ion usually has a single charge, the m/z ratio is usually the mass of the ion, or its molecular weight (abbreviated MW). Often, the terms m/z, the mass of the sample in Daltons (or molecular weight, abbreviated MW) are used interchangeably.
One way of measuring the mass of the sample accelerates the charged molecule, or ion, into a magnetic field. The sample ion moves under the influence of the magnetic field. A detector can be placed at the end of the path through the magnetic field, and the m/z of the molecule calculated as a function of the path through the magnetic field and the strength of the magnetic field.
Another method of measuring the mass of the sample is time-of-flight (TOF). TOF accelerates the sample ion with a known voltage, and measures how long it takes a sample ion, or the sample ion's fragments if the sample breaks down, to travel a known distance.
Yet another method, quadrupole mass analysis, rapidly alternates the magnetic polarities of pairs of magnetic poles permitting only sample molecules with a narrow range of masses to reach a detector.
Post source decay (PSD) studies are an extension of time-of-flight measurements. In a time-of-flight study, the sample can break into pieces, or fragment, after ionization and acceleration. When the sample fragments after it has been accelerated by the voltage, the resulting pieces, or fragments, all travel at the same speed, and therefore arrive at the detector at the same time as the unbroken sample would have arrived. The fragmentation of the sample can be studied by reflecting the sample ion with a repelling electric field. The reflected ions have different speeds that depend on their different masses. The mass of the reflected fragments can then be measured to better understand the molecular structure of the sample.
Molecules that are not easily rendered gaseous are more difficult to study with mass spectrometry. Accordingly, modern advances in mass spectroscopy often address problems regarding the handling of liquid or solid samples. When a molecule is `on` a substrate, the sample is adsorbed to that substrate. Desorption is the process by which a molecule adsorbed on a substrate is removed from the substrate. Removing a molecule from a surface is "desorbing" a molecule from that surface. Instead of starting with a gaseous sample, as basic mass spectrometry does, desorption mass spectrometry starts with the sample adsorbed on a substrate.
Desorption mass spectrometry has undergone significant improvements since the original experiments by Thomson were performed over ninety years ago. Thomson, Philosophical Magazine 20, 752 (1910).
The most dramatic change occurred in the early 1980's with the introduction of an organic matrix as a vehicle for desorbing and ionizing a sample. Liu, et al., Anal. Chem. 53, 109 (1981); Barber, et al., Nature 293, 270-275(1981); Karas, et al., Anal. Chem. 60, 2299-2301 (1988). Rather than using an electron beam to ionize a sample, MALDI ionizes a sample by transferring a proton from the organic matrix to the sample as part of the vaporization process. Although the electron beam ionization processes of the past can be useful for certain easily studied molecules, it is inadequate for modern studies. The development of proton transfer ionization has made biomolecular mass spectroscopy possible.
The broad success of matrix-assisted laser desorption/ionization (MALDI) is related to the ability of the matrix to incorporate and transfer energy to the sample. Barber, et al., Nature 293, 270-275 (1981); Karas, et al., Anal. Chem. 60, 2299-2301 (1988); Macfarlane, et al., Science 191, 920-925 (1976); Hillenkamp, et al., Anal. Chem. 63, A1193-A1202 (1991). For instance, in MALDI the sample is typically dissolved into a solid, ultraviolet-absorbing, crystalline organic acid matrix that vaporizes upon pulsed laser radiation, carrying the sample with the vaporized matrix. Karas, et al., Anal. Chem. 60, 2299-2301 (1988); Hillenkamp, et al., Anal. Chem. 63, A1193-A1202 (1991).
Direct desorption/ionization without a matrix has been extensively studied on a variety of substrates. For examples see: Zenobi, R. Chimia 51, 801-803 (1997); Zhan, et al., J. Am. Soc. Mass Spec. 8, 525-531 (1997); Hrubowchak, et al., Anal. Chem. 63, 1947-1953 (1991); Varakin, et al., High Energy Chemistry 28, 406-411 (1994); Wang, et al., Appl. Surf. Sci. 93, 205-210 (1996); and Posthumus, et al., Anal. Chem. 50, 985-991 (1978). Such procedures have not yet been widely used because of rapid molecular degradation and fragmentation usually observed upon direct exposure to laser radiation.
Further, salts and buffers can be detrimental to mass spectroscopy analyses. Biomolecular analysis in general and protein analysis in particular is subject to these limitations. Salts and buffers and can cause problems when only small quantities of sample are available, as sample can be lost in attempting to purify the sample. Moreover, salts normally form adduct peaks in a mass spectrum that compete with the peaks of the molecular ion dividing and broadening the overall signal. High pH value buffers can also interfere with ionization of the sample in MALDI or electrospray ionization (ESI) techniques.
ESI ionizes a sample by spraying and evaporating a highly electrically charged liquid containing the sample. ESI is sensitive to salts and buffers, with concentrations of salts and buffers over approximately one millimolar (mM) presenting problems. The common sodium and potassium ions in particular are a problem for ESI at concentrations above 10 mM. Although MALDI is not as sensitive to salts and buffers as ESI, MALDI is sensitive to salts and buffers, with concentrations of salts and buffers less than 10 mM being recommended for MALDI. Nevertheless, in MALDI, salts and buffers can interfere with the formation of the matrix crystal, and result in loss of signal.
MALDI is also severely limited in the study of small molecules. The MALDI matrix interferes with measurements below a m/z of approximately 700, called the low-mass region, which varies somewhat depending on the matrix used. Although MALDI-MS (matrix assisted laser ionization/desorption mass spectrometry) analysis can be utilized for small molecules as has been demonstrated by Lidgard, et al Rapid Comm. in Mass Spectrom. 9, 128-132 (1995) and matrix suppression can be achieved under certain circumstances as demonstrated by Knochenmuss, et al, Rapid Comm. in Mass Spectrom. 10, 871-877 (1996), matrix interference presents a real limitation on the study of the low-mass region via MALDI-MS. Siuzdak, Mass Spectrometry for Biotechnology, 162 (Academic Press, San Diego, 1996). Wang, et al, U.S. Pat. No. 5,869,832 recognize that there are few compounds that can form crystals that incorporate proteins, absorb light energy, and eject and ionize the protein intact.
Even with large molecules, MALDI has significant limitations. The matrix and matrix fragments can form adducts with the sample ion. The presence of adducts in a MALDI study can cause the measured signal to have a range of molecular weights. The range of molecular weights caused by the adducts results in a broadening the sample signal over a range of molecular weights. The broadening appears in a spectrum by the sample's peak height being substantially shortened when compared to the peak height of a non-broadened signal for the molecular ion of the sample.
In addition to the limitations MALDI has in studying molecules by direct measurements, MALDI is also limited in studying the Post Source Decay (PSD) of molecules. In MALDI, the vaporized matrix molecules of the sample interfere with the measurement of the fragments after reflection, rendering MALDI impractical even for molecules with a molecular weight over 700 Daltons.
Mass spectrometry is not the only field of study where generating ions is an important step for biomolecular analysis. Similar challenges are faced in electromagnetic spectroscopy of biomolecular ions.
Secondary ion mass spectrometry (SIMS) has had a profound effect on surface science as described by Benninghoven, et al., Secondary Ion Mass Spectrometry, 1227 (John Wiley & Sons, 1987). Indeed, U.S. Pat. No. 5,834,195 teaches that SIMS can be used to assay for the mass of an analyte that is covalently bonded to a substrate surface.
It would be beneficial to have a direct laser desorption/ionization technique for use in biomolecular and other analyses that addresses the needs still unfulfilled by the present methods for dramatically simplified sample preparation; i.e., an absence of a matrix or the need for covalent linkage of the analyte to the substrate, substrates tailored to the needs of a particular sample, and a tolerance for salts and buffers. The present invention addresses some of these needs highlighted by the limitations of current methods, and offers further benefits that are described herein.